The free radical theory of aging postulates that the accumulation of reactive oxygen and nitrogen species is causally linked to the progressive decline in the functional capacity of aging organisms. We have now developed the tools to test this hypothesis. We will detect, monitor and specify which oxidative stress conditions exist in aging organisms. We will accomplish this by utilizing the unique characteristics of oxidative protein thiol modifications: high sensitivity to the presence of small amounts of oxidants, high specificity to distinct reactive oxidants and reversibility both in vitro and in vivo. We are able to quantitatively describe changes in the thiol oxidation status of hundreds of proteins in a single experiment. This allows us to identify proteins that are significantly thiol-modified during chronological yeast aging and to define the type(s) of reactive oxygen or nitrogen species that develop during aging. Wild type and longevity mutants of S. cerevisiae will be used to investigate if a link exists between the onset, type(s) and extent of oxidative stress that cells encounter, and the life span of the organism. We will quantitatively describe oxidative protein modifications at early stages of yeast aging. This will reveal proteins that are particularly sensitive to early changes in the cellular redox status. The functional alteration of these proteins might be responsible for changes observed in metabolic and signaling pathways of chronologically aging yeast cells. We have developed fluorescent-based in vivo redox sensors that we will use to detect reactive oxygen species in S. cerevisiae and C. elegans. This is a very novel approach, which provides us with the unique opportunity to obtain a real-time picture of the oxidative stress conditions that develop in cells and tissues as an organism ages. The knowledge that we will gain from our studies could form the basis for the development of more specific antioxidants that combat these oxidants in vivo and possibly extend longevity.